Hematopoietic recovery of ex vivo perfusion culture expanded bone marrow and unexpanded peripheral blood progenitors after myeloablative chemotherapy

Abstract

Ex vivo culture of hematopoietic progenitor cells for autologous transplantation has generated world-wide interest, since it offers the prospect of using a limited cell number, and may allow more efficient gene transfer and passive elimination of contaminating tumor cells. In this study, we expanded bone marrow (BM) cells from 10 breast cancer patients to determine whether small BM aliquots can durably restore hematopoiesis, and whether thrombopoietin (TPO) improves hematopoietic reconstitution after myeloablative chemotherapy. We used the AastromReplicell System (ARS), performing a computer-controlled, stromal-based cell expansion process with frequent medium, cytokine and gas exchange. For the inoculation of 9 x 10(8) MNC, a median BM volume of 97.8 ml (range, 72.4-272) was harvested. We found a median 4.5-fold nucleated cell expansion, an 18-fold CFU-GM expansion, and 69% of input LTC-IC numbers. Nucleated and Lin-/CD34+ cells were infused with a median of 43.5 x 10(6)/kg (range, 34.1-71.7) and 2.8 x 10(5)/kg (range, 0.95-5.9), respectively. Despite tumor cell detection by immunocytochemical staining in 3/10 patients before expansion, tumor cells were not detectable in 9/10, and in one patient 1 log reduced post ARS culture. Following high-dose STAMP V chemotherapy, all patients received 12-day expanded BM cells. The median time to engraftment was 17 days (range, 11-20) for WBC >1000/microl, and 28 days (range, 21-55) for platelets >20,000/microl. A correlation between post-expansion Lin-/CD34+ cells and engraftment for ANC >500/microl, WBC >1000/microl and platelets >20,000/microl was observed. Hematopoiesis has been maintained for a median of 15 (range, 6-24) months. Our results demonstrate that transplantation of ex vivo expanded small BM aliquots allows hematopoietic reconstitution after myeloablative chemotherapy. Ex vivo generated ARS cells can reduce the risk of tumor cell reinoculation with autotransplants and may be valuable in settings in which only small stem cell doses are available, eg when using cord blood transplants or in non-mobilizing patients.

Clinical application of hematopoietic progenitor cell expansion: current status and future prospects

“…When used as the sole source of hematopoietic support, this graft reconstituted neutrophil count after a median 16 (range 13-24) days and platelet count at a median of 24 (range 19-45) days. Engelhardt et al 46 found almost precisely the same results in a trial of similar design; neutrophil nadirs ranged from 9 to 18 (median 16) days and platelet nadirs spanned 16-49 (median 23) days. These findings are similar to unexpanded bone marrow, but slower than MPB.…”

“…2,45 Two groups of investigators began to incorporate the stromal components into the expansion cultures. 44,46 These two studies examined whether small aliquots of steady-state bone marrow can be harvested, expanded ex vivo, and used as the sole source to support hematopoietic recovery after high-dose chemotherapy. Both studies utilized a perfusion-based stromal cellcontaining expansion device that employed frequent medium, cytokine, and gas exchange.…”

Ex vivo expansion of megakaryocyte precursor cells in autologous stem cell transplantation for relapsed malignant lymphoma

“…This observation is in concordance with others showing that there is a strong inverse correlation between the expanded cell dose and the time to neutrophil or platelet recovery. [17][18][19] In our study, this correlation could explain the fact that, despite a short median date of platelet transfusion independence, all patients received at least one platelet transfusion. It could therefore be proposed to perform a future ex vivo expansion trial of MK progenitors by a combination of MGDF þ SCF after starting the culture with a much higher number of CD34 þ cells (45 Â 10 6 /kg) than that used in our study (1-2 Â 10 6 CD34 þ /kg).…”

CD34+ or CD34−: which is the more primitive?

“…5 Apart from cell surface markers for isolation of HSC, numerous other assays that measure the stem cell activity have been exploited. [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] Clonal analyses using limiting dilution assays in Dexter-like cultures have been used (quality analysis), however, these are time-consuming due to the number of flasks that have to be analyzed. 26,27 In the past, HSC have been characterized with respect to their ability to form spleen colonies in irradiated mice (CFU-S).…”